Ultrastructural Analysis of Melanocyte Organelles

Control and Rab4A-knockdown melanocytes were seeded on Matrigel-coated glass coverslips. After 24 h, cells were fixed initially with 0.5% Karnovsky's fixative (4% paraformaldehyde, 72 mM sodium cacodylate pH 7.4, 4 mM CaCl₂, 0.5% glutaraldehyde) for 2 h followed by overnight fixation with 2% Karnovsky's fixative (contains 2% glutaraldehyde). Cells were processed for Epon embedding as described previously (Raposo et al., 2001 (link)). Ultrathin sections of cell monolayers were prepared with a Reichert UltracutS ultramicrotome (Leica Microsystems) and contrasted with uranyl acetate and lead citrate as described previously (Raposo et al., 2001 (link)). Samples were examined with a FEI Tecnai Spirit electron microscope (FEI Company), and digital acquisitions were made with a numeric camera (Quemesa; Soft Imaging System). For quantification, melanosome stages were defined by morphology (Raposo et al., 2001 (link)) and vacuoles were defined as empty organelles. The number of melanosomes and vacuoles per µm² cytosol were counted using ImageJ software. We counted ten cells from each control sh and Rab4A sh condition. Furthermore, we estimated the melanosome stages from 883 total melanosomes of control sh and 300 total melanosomes of Rab4A sh cells.